Rapid quantification and analysis of kinetic •OH radical footprinting data using SAFA.
Rapid quantification and analysis of kinetic •OH radical footprinting data using SAFA.
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DOI：
10.1016/s0076-6879(09)68003-4
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发表时间：
2009
影响因子：
--
通讯作者：
Laederach, Alain
中科院分区：
文献类型：
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作者：
Simmons, Katrina;Martin, Joshua S.;Shcherbakova, Inna;Laederach, Alain
使用高反应性化学物质来探测核酸的结构和动力学被软件大大简化，该软件能够快速定量从这些实验中产生的凝胶图像。SAFA（半自动足迹分析）允许用户通过一系列步骤快速和可重复地量化化学足迹凝胶图像，这些步骤包括校正、分配和整合相对条带强度。该程序的输出是报告每个核苷酸与化学探针的相对反应性的原始条带强度。我们在这里描述如何获得这些原始带强度使用SAFA和随后的标准化和分析程序需要处理这些数据。特别是，我们专注于分析时间分辨的羟基自由基（·OH）数据，我们用它来监测大RNA（L-21 T）折叠的动力学。嗜热菌I组内含子）。在折叠过程中的特定时间点，将RNA暴露于·OH自由基的爆发中，可以监测反应的时间进程。具体来说，我们确定了保护（折叠时无法接近·OH自由基探针的核苷酸）和不变（对·OH探针具有恒定可接近性的核苷酸）残基，我们将其用于数据的监测和标准化。通过这种分析，我们获得了时间进度曲线，从中我们确定折叠的动力学速率。我们还报告在SAFA中实现的数据可视化工具，允许用户将数据映射到二级结构图上。
The use of highly reactive chemical species to probe the structure and dynamics of nucleic acids is greatly simplified by software that enables rapid quantification of the gel images that result from these experiments. SAFA (Semi-Automated Footprinting Analysis) allows a user to quickly and reproducibly quantify a chemical footprinting gel image through a series of steps that rectify, assign, and integrate the relative band intensities. The output of this procedure is raw band intensities that report on the relative reactivity of each nucleotide with the chemical probe. We describe here how to obtain these raw band intensities using SAFA and the subsequent normalization and analysis procedures required to process this data. In particular, we focus on analyzing time-resolved hydroxyl radical (•OH) data, which we use to monitor the kinetics of folding of a large RNA (the L-21 T. thermophila group I intron). Exposing the RNA to bursts of •OH radicals at specific time-points during the folding process monitors the time-progress of the reaction. Specifically, we identify protected (nucleotides that become inaccessible to the •OH radical probe when folded) and invariant (nucleotides with constant accessibility to the •OH probe) residues that we use for monitoring and normalization of the data. With this analysis, we obtain time-progress curves from which we determine kinetic rates of folding. We also report on a data visualization tool implemented in SAFA that allows users to map data onto a secondary structure diagram.